Synchrotron tomography of a stem lizard elucidates early squamate anatomy.

Squamates (lizards and snakes) include more than 10,000 living species, descended from an ancestor that diverged more than 240 million years ago from that of their closest living relative, Sphenodon. However, a deficiency of fossil evidence1-7, combined with serious conflicts between molecular and morphological accounts of squamate phylogeny8-13 (but see ref. 14), has caused uncertainty about the origins and evolutionary assembly of squamate anatomy. Here we report the near-complete skeleton of a stem squamate, Bellairsia gracilis, from the Middle Jurassic epoch of Scotland, documented using high-resolution synchrotron phase-contrast tomography. Bellairsia shares numerous features of the crown group, including traits related to cranial kinesis (an important functional feature of many extant squamates) and those of the braincase and shoulder girdle. Alongside these derived traits, Bellairsia also retains inferred ancestral features including a pterygoid-vomer contact and the presence of both cervical and dorsal intercentra. Phylogenetic analyses return strong support for Bellairsia as a stem squamate, suggesting that several features that it shares with extant gekkotans are plesiomorphies, consistent with the molecular phylogenetic hypothesis that gekkotans are early-diverging squamates. We also provide confident support of stem squamate affinities for the enigmatic Oculudentavis. Our findings indicate that squamate-like functional features of the suspensorium, braincase and shoulder girdle preceded the origin of their palatal and vertebral traits and indicate the presence of advanced stem squamates as persistent components of terrestrial assemblages up to at least the middle of the Cretaceous period.

Fossilized cell structures identify an ancient origin for the teleost whole-genome duplication.

Teleost fishes comprise one-half of all vertebrate species and possess a duplicated genome. This whole-genome duplication (WGD) occurred on the teleost stem lineage in an ancient common ancestor of all living teleosts and is hypothesized as a trigger of their exceptional evolutionary radiation. Genomic and phylogenetic data indicate that WGD occurred in the Mesozoic after the divergence of teleosts from their closest living relatives but before the origin of the extant teleost groups. However, these approaches cannot pinpoint WGD among the many extinct groups that populate this 50- to 100-million-y lineage, preventing tests of the evolutionary effects of WGD. We infer patterns of genome size evolution in fossil stem-group teleosts using high-resolution synchrotron X-ray tomography to measure the bone cell volumes, which correlate with genome size in living species. Our findings indicate that WGD occurred very early on the teleost stem lineage and that all extinct stem-group teleosts known so far possessed duplicated genomes. WGD therefore predates both the origin of proposed key innovations of the teleost skeleton and the onset of substantial morphological diversification in the clade. Moreover, the early occurrence of WGD allowed considerable time for postduplication reorganization prior to the origin of the teleost crown group. This suggests at most an indirect link between WGD and evolutionary success, with broad implications for the relationship between genomic architecture and large-scale evolutionary patterns in the vertebrate Tree of Life.

Testing heterochrony: Connecting skull shape ontogeny and evolution of feeding adaptations in baleen whales.

Ontogeny plays a key role in the evolution of organisms, as changes during the complex processes of development can allow for new traits to arise. Identifying changes in ontogenetic allometry-the relationship between skull shape and size during growth-can reveal the processes underlying major evolutionary transformations. Baleen whales (Mysticeti, Cetacea) underwent major morphological changes in transitioning from their ancestral raptorial feeding mode to the three specialized filter-feeding modes observed in extant taxa. Heterochronic processes have been implicated in the evolution of these feeding modes, and their associated specialized cranial morphologies, but their role has never been tested with quantitative data. Here, we quantified skull shapes ontogeny and reconstructed ancestral allometric trajectories using 3D geometric morphometrics and phylogenetic comparative methods on sample representing modern mysticetes diversity. Our results demonstrate that Mysticeti, while having a common developmental trajectory, present distinct cranial shapes from early in their ontogeny corresponding to their different feeding ecologies. Size is the main driver of shape disparity across mysticetes. Disparate heterochronic processes are evident in the evolution of the group: skim feeders present accelerated growth relative to the ancestral nodes, while Balaenopteridae have overall slower growth, or pedomorphosis. Gray whales are the only taxon with a relatively faster rate of growth in this group, which might be connected to its unique benthic feeding strategy. Reconstructed ancestral allometries and related skull shapes indicate that extinct taxa used less specialized filter-feeding modes, a finding broadly in line with the available fossil evidence.

Ontogenetic variability of the intertympanic sinus distinguishes lineages within Crocodylia.

The phylogenetic relationships within crown Crocodylia remain contentious due to conflicts between molecular and morphological hypotheses. However, morphology-based datasets are mostly constructed on external characters, overlooking internal structures. Here, we use 3D geometric morphometrics to study the shape of the intertympanic sinus system in crown crocodylians during ontogeny, in order to assess its significance in a taxonomic context. Intertympanic sinus shape was found to be highly correlated with size and modulated by cranial shape during development. Still, adult sinus morphology distinguishes specimens at the family, genus and species level. We observe a clear distinction between Alligatoridae and Longirostres, a separation of different Crocodylus species and the subfossil Malagasy genus Voay, and a distinction between the Tomistoma and Gavialis lineages. Our approach is independent of molecular methods but concurs with the molecular topologies. Therefore, sinus characters could add significantly to morphological datasets, offering an alternative viewpoint to resolve problems in crocodylian relationships.

Synchrotron scanning reveals amphibious ecomorphology in a new clade of bird-like dinosaurs.

Maniraptora includes birds and their closest relatives among theropod dinosaurs. During the Cretaceous period, several maniraptoran lineages diverged from the ancestral coelurosaurian bauplan and evolved novel ecomorphologies, including active flight, gigantism, cursoriality and herbivory. Propagation X-ray phase-contrast synchrotron microtomography of a well-preserved maniraptoran from Mongolia, still partially embedded in the rock matrix, revealed a mosaic of features, most of them absent among non-avian maniraptorans but shared by reptilian and avian groups with aquatic or semiaquatic ecologies. This new theropod, Halszkaraptor escuilliei gen. et sp. nov., is related to other enigmatic Late Cretaceous maniraptorans from Mongolia in a novel clade at the root of Dromaeosauridae. This lineage adds an amphibious ecomorphology to those evolved by maniraptorans: it acquired a predatory mode that relied mainly on neck hyperelongation for food procurement, it coupled the obligatory bipedalism of theropods with forelimb proportions that may support a swimming function, and it developed postural adaptations convergent with short-tailed birds.

The ontogeny of asymmetry in echolocating whales.

Extreme asymmetry of the skull is one of the most distinctive traits that characterizes toothed whales (Odontoceti, Cetacea). The origin and function of cranial asymmetry are connected to the evolution of echolocation, the ability to use high-frequency sounds to navigate the surrounding environment. Although this novel phenotype must arise through changes in cranial development, the ontogeny of cetacean asymmetry has never been investigated. Here we use three-dimensional geometric morphometrics to quantify the changes in degree of asymmetry and skull shape during prenatal and postnatal ontogeny for five genera spanning odontocete diversity (oceanic dolphins, porpoises and beluga). Asymmetry in early ontogeny starts low and tracks phylogenetic relatedness of taxa. Distantly related taxa that share aspects of their ecology overwrite these initial differences via heterochronic shifts, ultimately converging on comparable high levels of skull asymmetry. Porpoises maintain low levels of asymmetry into maturity and present a decelerated rate of growth, probably retained from the ancestral condition. Ancestral state reconstruction of allometric trajectories demonstrates that both paedomorphism and peramorphism contribute to cranial shape diversity across odontocetes. This study provides a striking example of how divergent developmental pathways can produce convergent ecological adaptations, even for some of the most unusual phenotypes exhibited among vertebrates.

A reassessment of the enigmatic diapsid Paliguana whitei and the early history of Lepidosauromorpha.

Lepidosaurs include lizards, snakes, amphisbaenians and the tuatara, comprising a highly speciose evolutionary radiation with widely varying anatomical traits. Their stem-lineage originated by the late middle Permian 259 million years ago, but its early fossil record is poorly documented, obscuring the origins of key anatomical and functional traits of the group. Paliguana whitei, from the Early Triassic of South Africa, is an enigmatic fossil species with the potential to provide information on this. However, its anatomy and phylogenetic affinities remain highly uncertain, and have been debated since its discovery more than 100 years ago. We present microtomographic three-dimensional imaging of the cranial anatomy of P. whitei that clarifies these uncertainties, providing strong evidence for lepidosauromorph affinities based on the structure of the temporal region and the implantation of marginal dentition. Phylogenetic analysis including these new data recovers Paliguana as the earliest known stem-lepidosaur, within a long-lived group of early diverging lepidosauromorphs that persisted to at least the Middle Jurassic. Our results provide insights into cranial evolution on the lepidosaur stem-lineage, confirming that characteristics of pleurodont dental implantation evolved early on the lepidosaur stem-lineage. By contrast, key functional traits related to hearing (quadrate conch) and feeding (streptostyly) evolved later in the lepidosaur crown-group.

Functional constraints during development limit jaw shape evolution in marsupials.

Differences in jaw function experienced through ontogeny can have striking consequences for evolutionary outcomes, as has been suggested for the major clades of mammals. By contrast to placentals, marsupial newborns have an accelerated development of the head and forelimbs, allowing them to crawl to the mother's teats to suckle within just a few weeks of conception. The different functional requirements that marsupial newborns experience in early postnatal development have been hypothesized to have constrained their morphological diversification relative to placentals. Here, we test whether marsupials have a lower ecomorphological diversity and rate of evolution in comparison with placentals, focusing specifically on their jaws. To do so, a geometric morphometric approach was used to characterize jaw shape for 151 living and extinct species of mammals spanning a wide phylogenetic, developmental and functional diversity. Our results demonstrate that jaw shape is significantly influenced by both reproductive mode and diet, with substantial ecomorphological convergence between metatherians and eutherians. However, metatherians have markedly lower disparity and rate of mandible shape evolution than observed for eutherians. Thus, despite their ecomorphological diversity and numerous convergences with eutherians, the evolution of the jaw in metatherians appears to be strongly constrained by their specialized reproductive biology.

Gaseous environment modulates volatile emission and viability loss during seed artificial ageing.

MAIN CONCLUSION: Modulation of the gaseous environment using oxygen absorbers and/or silica gel shows potential for enhancing seed longevity through trapping toxic volatiles emitted by seeds during artificial ageing. Volatile profiling using non-invasive gas chromatography-mass spectrometry provides insight into the specific processes occurring during seed ageing. Production of alcohols, aldehydes and ketones, derived from processes such as alcoholic fermentation, lipid peroxidation and Maillard reactions, are known to be dependent on storage temperature and relative humidity, but little is known about the potential modulating role of the gaseous environment, which also affects seed lifespan, on volatile production. Seeds of Lolium perenne (Poaceae), Agrostemma githago (Caryophyllaceae) and Pisum sativum (Fabaceae) were aged under normal atmospheric oxygen conditions and in sealed vials containing either oxygen absorbers, oxygen absorbers and silica gel (equilibrated at 60% RH), or silica gel alone. Seeds of A. githago that were aged in the absence of oxygen maintained higher viability and produced fewer volatiles than seeds aged in air. In addition, seeds of A. githago and L. perenne aged in the presence of silica gel were longer lived than those aged without silica, with no effect on seed moisture content or oxygen concentration in the storage containers, but with silica gel acting as a volatile trap. These results indicate that the use of inexpensive oxygen absorbers and silica gel could improve seed longevity in storage for some species and suggests a potential, and previously unidentified, role for silica gel in ultra-dry storage.

Ontogenetic development of the holocephalan dentition: Morphological transitions of dentine in the absence of teeth.

Among the cartilaginous fishes (Chondrichthyes), the Holocephali are unique in that teeth are absent both in ontogeny and adult regenerative growth. Instead, the holocephalan dentition of ever-growing nonshedding dental plates is composed of dentine, trabecular in arrangement, forming spaces into which a novel hypermineralized dentine (whitlockin) is deposited. These tissue features form a variety of specific morphologies as the defining characters of dental plates in the three families of extant holocephalans. We demonstrate how this morphology changes through ontogenetic development with continuity between morphologies, through successive growth stages of the dentition represented by the dental plate. For example, rod-shaped whitlockin appears early, later transformed into the tritoral pad, including a regular arrangement of vascular canals and whitlockin forming with increasing mineralization (95%-98%). While the tritoral pads develop lingually, stacks of individual ovoids of whitlockin replace the rods in the more labial parts of the plate, again shaped by the forming trabecular dentine. The ability to make dentine into new, distinctive patterns is retained in the evolution of the Holocephali, despite the lack of teeth forming in development of the dentition. We propose that developmentally, odontogenic stem cells, retained through evolution, control the trabecular dentine formation within the dental plate, and transition to form whitlockin, throughout lifetime growth. Our model of cellular activity proposes a tight membrane of odontoblasts, having transformed to whitloblasts, that can control active influx of minerals to the rapidly mineralizing dentine, forming whitlockin. After the reduced whitloblast cells transition back to odontoblasts, they continue to monitor the levels of minerals (calcium, phosphate and magnesium) and at a slower rate of growth in the peritubate 'softer' dentine. This model explains the unique features of transitions within the holocephalan dental plate morphology.

Paleohistology of the Cretaceous resin-producing conifer Geinitzia reichenbachii using X-ray synchrotron microtomography.

PREMISE: The conifer Geinitzia reichenbachii was a common member of the Cretaceous Laurasian floras. However, the histology of G. reichenbachii leafy axes was never described in detail, and our knowledge of its paleoecology remains very limited. Using new and exquisitely preserved silicified material from the Upper Cretaceous of western France, we describe G. reichenbachii from the gross morphology to the cellular scale, then discuss paleoecological and taphonomical implications. METHODS: We examined specimens from two localities in western France (Claix and Moragne) using propagation phase-contrast X-ray synchrotron microtomography. RESULTS: The cuticle and the inner tissues of leafy axes are preserved in three dimensions. Epidermis, hypodermis, palisade parenchyma, spongy parenchyma, transfusion tracheids, and most of tissues of the vascular cylinder are clearly discernible. The numerous resin ducts are sometimes filled by persisting resin. Additionally, surfaces of some leaves preserved drops and flows of resin. CONCLUSIONS: Depositional environmental context combined with histological features of G. reichenbachii suggest that this conifer was adapted to a range of marginal-littoral ecosystems including those open to the sea paleoenvironments and innermost ones influenced by strong continental inputs. Geinitzia reichenbachii was adapted to withstand intense sunlight, hot temperatures coupled with salty sea wind, and dry conditions. The frequent amber-bearing beds in the Cretaceous from western France with Geinitzia as a main component of the associated floras raises the possibility of a role for Geinitzia in the production of the "Charentese amber".

The Often-Overlooked Power of Summary Statistics in Exploratory Data Analysis: Comparison of Pattern Recognition Entropy (PRE) to Other Summary Statistics and Introduction of Divided Spectrum-PRE (DS-PRE).

Unsupervised exploratory data analysis (EDA) is often the first step in understanding complex data sets. While summary statistics are among the most efficient and convenient tools for exploring and describing sets of data, they are often overlooked in EDA. In this paper, we show multiple case studies that compare the performance, including clustering, of a series of summary statistics in EDA. The summary statistics considered here are pattern recognition entropy (PRE), the mean, standard deviation (STD), 1-norm, range, sum of squares (SSQ), and X4, which are compared with principal component analysis (PCA), multivariate curve resolution (MCR), and/or cluster analysis. PRE and the other summary statistics are direct methods for analyzing data-they are not factor-based approaches. To quantify the performance of summary statistics, we use the concept of the "critical pair," which is employed in chromatography. The data analyzed here come from different analytical methods. Hyperspectral images, including one of a biological material, are also analyzed. In general, PRE outperforms the other summary statistics, especially in image analysis, although a suite of summary statistics is useful in exploring complex data sets. While PRE results were generally comparable to those from PCA and MCR, PRE is easier to apply. For example, there is no need to determine the number of factors that describe a data set. Finally, we introduce the concept of divided spectrum-PRE (DS-PRE) as a new EDA method. DS-PRE increases the discrimination power of PRE. We also show that DS-PRE can be used to provide the inputs for the k-nearest neighbor (kNN) algorithm. We recommend PRE and DS-PRE as rapid new tools for unsupervised EDA.

Three-dimensional characterization of osteocyte volumes at multiple scales, and its relationship with bone biology and genome evolution in ray-finned fishes.

Osteocytes, cells embedded within the bone mineral matrix, inform on key aspects of vertebrate biology. In particular, a relationship between volumes of the osteocytes and bone growth and/or genome size has been proposed for several tetrapod lineages. However, the variation in osteocyte volume across different scales is poorly characterized and mostly relies on incomplete, two-dimensional information. In this study, we characterize the variation of osteocyte volumes in ray-finned fishes (Actinopterygii), a clade including more than half of modern vertebrate species in which osteocyte biology is poorly known. We use X-ray synchrotron micro-computed tomography (SRµCT) to achieve a three-dimensional visualization of osteocyte lacunae and direct measurement of their size (volumes). Our specimen sample is designed to characterize variation in osteocyte lacuna morphology at three scales: within a bone, among the bones of one individual and among species. At the intra-bone scale, we find that osteocyte lacunae vary noticeably in size between zones of organized and woven bone (being up to six times larger in woven bone), and across cyclical bone deposition. This is probably explained by differences in bone deposition rate, with larger osteocyte lacunae contained in bone that deposits faster. Osteocyte lacuna volumes vary 3.5-fold among the bones of an individual, and this cannot readily be explained by variation in bone growth rate or other currently observable factors. Finally, we find that genome size provides the best explanation of variation in osteocyte lacuna volume among species: actinopterygian taxa with larger genomes (polyploid taxa in particular) have larger osteocyte lacunae (with a ninefold variation in median osteocyte volume being measured). Our findings corroborate previous two-dimensional studies in tetrapods that also observed similar patterns of intra-individual variation and found a correlation with genome size. This opens new perspectives for further studies on bone evolution, physiology and palaeogenomics in actinopterygians, and vertebrates as a whole.

Versailles Project on Advanced Materials and Standards interlaboratory study on intensity calibration for x-ray photoelectron spectroscopy instruments using low-density polyethylene.

We report the results of a Versailles Project on Advanced Materials and Standards interlaboratory study on the intensity scale calibration of x-ray photoelectron spectrometers using low-density polyethylene (LDPE) as an alternative material to gold, silver, and copper. An improved set of LDPE reference spectra, corrected for different instrument geometries using a quartz-monochromated Al Kα x-ray source, was developed using data provided by participants in this study. Using these new reference spectra, a transmission function was calculated for each dataset that participants provided. When compared to a similar calibration procedure using the NPL reference spectra for gold, the LDPE intensity calibration method achieves an absolute offset of ∼3.0% and a systematic deviation of ±6.5% on average across all participants. For spectra recorded at high pass energies (≥90 eV), values of absolute offset and systematic deviation are ∼5.8% and ±5.7%, respectively, whereas for spectra collected at lower pass energies (<90 eV), values of absolute offset and systematic deviation are ∼4.9% and ±8.8%, respectively; low pass energy spectra perform worse than the global average, in terms of systematic deviations, due to diminished count rates and signal-to-noise ratio. Differences in absolute offset are attributed to the surface roughness of the LDPE induced by sample preparation. We further assess the usability of LDPE as a secondary reference material and comment on its performance in the presence of issues such as variable dark noise, x-ray warm up times, inaccuracy at low count rates, and underlying spectrometer problems. In response to participant feedback and the results of the study, we provide an updated LDPE intensity calibration protocol to address the issues highlighted in the interlaboratory study. We also comment on the lack of implementation of a consistent and traceable intensity calibration method across the community of x-ray photoelectron spectroscopy (XPS) users and, therefore, propose a route to achieving this with the assistance of instrument manufacturers, metrology laboratories, and experts leading to an international standard for XPS intensity scale calibration.

The mystery of a missing bone: revealing the orbitosphenoid in basal Epicynodontia (Cynodontia, Therapsida) through computed tomography.

The basal non-mammaliaform cynodonts from the late Permian (Lopingian) and Early Triassic are a major source of information for the understanding of the evolutionary origin of mammals. Detailed knowledge of their anatomy is critical for understanding the phylogenetic transition toward mammalness and the paleobiological reconstruction of mammalian precursors. Using micro-computed tomography (µCT), we describe the internal morphology of the interorbital region that includes the rarely fossilized orbitosphenoid elements in four basal cynodonts. These paired bones, which are positioned relatively dorsally in the skull, contribute to the wall of the anterior part of the braincase and form the floor for the olfactory lobes. Unlike procynosuchids and the more basal therapsids in which the orbitosphenoids are well developed, dense, and bear a ventral keel, the basal epicynodonts Cynosaurus, Galesaurus, and Thrinaxodon display cancellous, reduced, and loosely articulated orbitosphenoids, a condition shared with many eucynodonts. The hemi-cylindrical orbitosphenoid from which the mammalian condition is derived re-evolved convergently in traversodontid and some probainognathian cynodonts.

Endocranial Casts of Pre-Mammalian Therapsids Reveal an Unexpected Neurological Diversity at the Deep Evolutionary Root of Mammals.

The origin and evolution of the mammalian brain has long been the focus of scientific enquiry. Conversely, little research has focused on the palaeoneurology of the stem group of Mammaliaformes, the Permian and Triassic non-mammaliaform Therapsida (NMT). This is because the majority of the NMT have a non-ossified braincase, making the study of their endocranial cast (sometimes called the "fossil brain") problematic. Thus, descriptions of the morphology and size of NMT endocranial casts have been based largely on approximations rather than reliable determination. Accordingly, here we use micro-CT scans of the skulls of 1 Dinocephalia and 3 Biarmosuchia, which are NMT with a fully ossified braincase and thus a complete endocast. For the first time, our work enables the accurate determination of endocranial shape and size in NMT. This study suggests that NMT brain size falls in the upper range of the reptilian and amphibian variation. Brain size in the dicynodont Kawingasaurus is equivalent to that of early Mammaliaformes, whereas the Dinocephalia show evidence of a secondary reduction of brain size. In addition, unlike other NMT in which the endocast has a tubular shape and its parts are arranged in a linear manner, the biarmosuchian endocast is strongly flexed at the level of the midbrain, creating a near right angle between the fore- and hindbrain. These data highlight an unexpected diversity of endocranial size and morphology in NMT, features that are usually considered conservative in this group.

Lead- and Iodide-Deficient (CH3 NH3 )PbI3 (d-MAPI): The Bridge between 2D and 3D Hybrid Perovskites.

3D and 2D hybrid perovskites, which have been known for more than 20 years, have emerged recently as promising materials for optoelectronic applications, particularly the 3D compound (CH3 NH3 )PbI3 (MAPI). The discovery of a new family of hybrid perovskites called d-MAPI is reported: the association of PbI2 with both methyl ammonium (MA+ ) and hydroxyethyl ammonium (HEA+ ) cations leads to a series of five compounds with general formulation (MA)1-2.48x (HEA)3.48x [Pb1-x I3-x ]. These materials, which are lead- and iodide-deficient compared to MAPI while retaining 3D architecture, can be considered as a bridge between the 2D and 3D materials. Moreover, they can be prepared as crystallized thin films by spin-coating. These new 3D materials appear very promising for optoelectronic applications, not only because of their reduced lead content, but also in account of the large flexibility of their chemical composition through potential substitutions of MA+ , HEA+ , Pb2+ and I- ions.

Morphometric analysis of chameleon fossil fragments from the Early Pliocene of South Africa: a new piece of the chamaeleonid history.

The evolutionary history of chameleons has been predominantly studied through phylogenetic approaches as the fossil register of chameleons is limited and fragmented. The poor state of preservation of these fossils has moreover led to the origin of numerous nomen dubia, and the identification of many chameleon fossils remains uncertain. We here examine chameleon fossil fragments from the Early Pliocene Varswater formation, exposed at the locality of Langebaanweg "E" Quarry along the southwestern coast of South Africa. Our aim was to explore whether these fossil fragments could be assigned to extant genera. To do so, we used geometric morphometric approaches based on microtomographic imaging of extant chameleons as well as the fossil fragments themselves. Our study suggests that the fossils from this deposit most likely represent at least two different forms that may belong to different genera. Most fragments are phenotypically dissimilar from the South African endemic genus Bradypodion and are more similar to other chameleon genera such as Trioceros or Kinyongia. However, close phenetic similarities between some of the fragments and the Seychelles endemic Archaius or the Madagascan genus Furcifer suggest that some of these fragments may not contain enough genus-specific information to allow correct identification. Other fragments such as the parietal fragments appear to contain more genus-specific information, however. Although our data suggest that the fossil diversity of chameleons in South Africa was potentially greater than it is today, this remains to be verified based on other and more complete fragments.

Neuroanatomy and pneumaticity of the extinct Malagasy "horned" crocodile Voay robustus and its implications for crocodylid phylogeny and palaeoecology.

Voay robustus, the extinct Malagasy "horned" crocodile, was originally considered to be the only crocodylian representative in Madagascar during most part of the Holocene. However, Malagasy crocodylian remains have had confused taxonomic attributions and recent studies have underlined that Crocodylus and Voay populations coexisted on the island for at least 7500 years. Here, we describe the inner braincase anatomy of Voay robustus using x-ray computed tomography on four specimens, to provide new anatomical information that distinguishes Voay from Crocodylus, especially features of the brain endocast and the paratympanic sinuses. Geometric morphometric analyses are performed on 3D models of the internal organs to compare statistically Voay with a subset of extant Crocodylidae. Following these comparisons, we build an endocranial morphological matrix to discuss the proposed phylogenetic affinities of Voay with Osteolaeminae from an endocranial point of view. Additionally, we discuss the use of internal characters in systematic studies and find that they can have a major impact on morphological analyses. Finally, new radiocarbon data on Voay and subfossil Crocodylus specimens are recovered between 2010 and 2750 cal BP, which confirm the cohabitation of the two species in the same area for a long period of time. We thus assess several extinction scenarios, and propose a slightly different ecology of Voay compared to Crocodylus, which could have allowed habitat partitioning on the island. Our approach complements information obtained from previous molecular and morphological phylogenies, as well as previous radiocarbon dating, together revealing past diversity and faunal turnovers in Madagascar.